Biophysical Cues and Corneal Wound Healing

生物物理线索和角膜伤口愈合

• 批准号: 9185333
• 负责人: CHRISTOPHER John MURPHY
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9185333
• 关键词: Actins; Address; Adhesions; Atomic Force Microscopy; Behavior; Biochemical; Biomechanics; Biophysics; Blindness; Cells; Chemistry; Coculture Techniques; Communication; Complex; Cornea; Corneal Injury; Corneal Opacity; Corneal Stroma; Cues; Cytoskeleton; Data; Drug usage; Engineering; Environment; Epithelial; Epithelial Cells; Evolution; Extracellular Matrix; Fiber; Fibroblasts; Fibrosis; Funding; Goals; Homeostasis; Hydrogels; In Vitro; Infection; Investigation; Knowledge; Laboratories; Laser In Situ Keratomileusis; Maintenance; Measures; Mechanics; Methods; Microfluidic Microchips; Microfluidics; Modeling; Myofibroblast; Operative Surgical Procedures; Oryctolagus cuniculus; Outcome; Patient-Focused Outcomes; Phenotype; Process; Production; Research; Research Personnel; Signal Transduction; Signaling Molecule; Stimulus; Stromal Cells; System; Testing; Therapeutic; Therapeutic Effect; Time; Tissues; Traction; Transforming Growth Factor beta; Trauma; Visual Acuity; Visual impairment; Wound Healing; cell behavior; cell type; clinically relevant; corneal epithelium; crosslink; design; experimental study; improved; in vivo; intervention effect; mechanical properties; migration; novel; public health relevance; repaired; response to injury; wound

 DESCRIPTION (provided by applicant): Corneal opacities, a leading cause of blindness worldwide, are commonly a result of stromal fibrosis or haze from dysregulated wound healing. Corneal wound healing is an enormously complex process that requires the simultaneous cellular integration of multiple soluble biochemical as well as biophysical cues associated with the wound space. Research from our laboratory and others have demonstrated biophysical attributes of the extracellular matrix profoundly modulate a host of fundamental cellular behaviors essential to the maintenance of homeostasis and wound repair including adhesion, migration, proliferation and differentiation. We have demonstrated that the intrinsic mechanical properties of the corneal stroma are significantly altered in a time dependent manner throughout wound healing. The matrix associated with the wound space was stiffest at 7 days with the greatest myofibroblast numbers and degree of stromal haze occurring at a later time point. This temporal relationship, combined with in vitro data documenting stiffer substrates to promote myofibroblast transformation, suggests a causal relationship. In Aim 1 we propose to directly modulate matrix stiffness in vivo using clinically relevant approaches and establish the impact of stiffening and softening of the wound matrix on wound healing outcomes using a well-defined rabbit PTK model. The complex inter-relationship between cytoskeletal dynamics and matrix remodeling and stiffness is central to determining wound healing outcomes but remains little investigated and poorly understood and are the central focus of Aim 2. Here, the impact of altering cytoskeletal dynamics in vitro on cell derived extracellular matrix elaboration, remodeling and mechanics, and the impact of altering cell derived matrix stiffness on keratocytes to myofibroblast transformation will be determined. Preliminary data demonstrate that the mechanical properties of ECM derived from cell in vitro can be modulated by applying diverse clinically relevant crosslinking methods. Preliminary data also document that modulating the cellular stiffness using drugs that alter the cytoskeleton can dramatically influence myofibroblast transformation. Stiffening of the cell promoted myofibroblast transformation. While softening of the cell inhibited transformation even in the presence of TGFβ signaling. Additionally, the complex signaling dynamic that exist between stromal and epithelial cells will be investigated using a novel microfluidic co-culture platform in Aim 3. This novel system allows for the simultaneous presentation of multiple yet distinct biophysical and biochemical stimuli in addition to controlled exchange of soluble signaling molecules between epithelial and stromal cells. We will determine if soluble signals from the epithelial cells have a differential effect on KFM transformation under various biophysical stimuli. In aggregate the experiments detailed in this proposal are aimed at testing our central concept that optimal outcomes in corneal wound repair can be facilitated by engineering the intrinsic biophysical attributes of cells and/or matrices.

 描述（由申请人提供）：角膜混浊是全球范围内导致失明的主要原因，通常是伤口愈合失调引起的基质纤维化或混浊的结果。角膜伤口愈合是一个非常复杂的过程，需要同时细胞整合多种可溶性生物化学以及与伤口空间相关的生物物理线索。来自我们实验室和其他人的研究已经证明细胞外基质的生物物理属性深刻地调节对维持稳态和伤口修复至关重要的许多基本细胞行为，包括粘附、迁移、增殖和分化。我们已经证明，在整个伤口愈合过程中，角膜基质的内在机械性能以时间依赖性方式显著改变。与伤口空间相关的基质在第7天时最硬，肌成纤维细胞数量和基质混浊程度最大，发生在稍后的时间点。这种时间关系，结合体外数据记录更硬的基板，以促进肌成纤维细胞转化，表明因果关系。在目标1中，我们提出使用临床相关方法直接调节体内基质硬度，并使用定义明确的兔PTK模型确定伤口基质硬化和软化对伤口愈合结果的影响。细胞骨架动力学与基质重塑和硬度之间的复杂相互关系对于确定伤口愈合结果至关重要，但仍然很少研究和了解，并且是目标2的中心焦点。在此，将确定改变细胞骨架动力学在体外对细胞衍生的细胞外基质加工、重塑和力学的影响，以及改变细胞衍生的基质硬度对角膜细胞向肌成纤维细胞转化的影响。初步数据表明，来自体外细胞的ECM的机械性能可以通过应用不同的临床相关的交联方法来调节。初步数据还证明，使用改变细胞骨架的药物调节细胞刚度可以显著影响肌成纤维细胞转化。细胞变硬促进肌成纤维细胞转化。而软化的细胞抑制转化，即使在TGFβ信号的存在下。此外，将使用Aim 3中的新型微流体共培养平台研究基质细胞和上皮细胞之间存在的复杂信号传导动力学。这种新的系统允许同时呈现多种不同的生物物理和生化刺激，除了上皮细胞和基质细胞之间的可溶性信号分子的受控交换。我们将确定来自上皮细胞的可溶性信号是否对 KFM在各种生物物理刺激下的转化。总的来说，本提案中详细描述的实验旨在测试我们的中心概念，即通过工程化细胞和/或基质的固有生物物理属性可以促进角膜伤口修复的最佳结果。